Systemic AAV vectors for widespread and targeted gene delivery in rodents

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing

Development of the mammalian cortical hem and its derivatives: the choroid plexus, Cajal–Retzius cells and hippocampus

The dorsal medial region of the developing mammalian telencephalon plays a central role in the patterning of the adjacent brain regions. This review describes the development of this specialized region of the vertebrate brain, called the cortical hem, and the formation of the various cells and structures it gives rise to, including the choroid plexus, Cajal–Retzius cells and the hippocampus. We highlight the ontogenic processes that create these different forebrain derivatives from their shared embryonic origin and discuss the key signalling pathways and molecules that influence the patterning of the cortical hem. These include BMP, Wnt, FGF and Shh signalling pathways acting with Homeobox factors to carve the medial telencephalon into district progenitor regions, which in turn give rise to the choroid plexus, dentate gyrus and hippocampus. We then link the formation of the lateral ventricle choroid plexus with embryonic and postnatal neurogenesis in the hippocampus

Cranial Nerve Development Requires Co-Ordinated Shh and Canonical Wnt Signaling

International audienceCranial nerves govern sensory and motor information exchange between the brain and tissues of the head and neck. The cranial nerves are derived from two specialized populations of cells, cranial neural crest cells and ectodermal placode cells. Defects in either cell type can result in cranial nerve developmental defects. Although several signaling pathways are known to regulate cranial nerve formation our understanding of how intercellular signaling between neural crest cells and placode cells is coordinated during cranial ganglia morpho-genesis is poorly understood. Sonic Hedgehog (Shh) signaling is one key pathway that regulates multiple aspects of craniofacial development, but whether it coordinates cranial neural crest cell and placodal cell interactions during cranial ganglia formation remains unclear. In this study we examined a new Patched1 (Ptch1) loss-of-function mouse mutant and characterized the role of Ptch1 in regulating Shh signaling during cranial ganglia development. Ptch1 Wig/ Wig mutants exhibit elevated Shh signaling in concert with disorganization of the trigeminal and facial nerves. Importantly, we discovered that enhanced Shh signaling suppressed canonical Wnt signaling in the cranial nerve region. This critically affected the survival and migration of cranial neural crest cells and the development of placodal cells as well as the integration between neural crest and placodes. Collectively, our findings highlight a novel and critical role for Shh signaling in cranial nerve development via the cross regulation of canonical Wnt signaling

Cux2 functions downstream of Notch signaling to regulate dorsal interneuron formation in the spinal cord

Obtaining the diversity of interneuron subtypes in their appropriate numbers requires the orchestrated integration of progenitor proliferation with the regulation of differentiation. Here we demonstrate through loss-of-function studies in mice that the Cut homeodomain transcription factor Cux2 (Cutl2) plays an important role in regulating the formation of dorsal spinal cord interneurons. Furthermore, we show that Notch regulates Cux2 expression. Although Notch signaling can be inhibitory to the expression of proneural genes, it is also required for interneuron formation during spinal cord development. Our findings suggest that Cux2 might mediate some of the effects of Notch signaling on interneuron formation. Together with the requirement for Cux2 in cell cycle progression, our work highlights the mechanistic complexity in balancing neural progenitor maintenance and differentiation during spinal cord neurogenesis

Increased Shh signaling in <i>Ptch1</i>^<i>Wig</i> mutants resulted in less cellular interaction between neural crest and placodal cells.

<p>(A-D) <i>Wnt1Cre;R26RYFP</i> fate mapping in control wild type (A and B) and <i>Ptch1</i>^{<i>Wig/Wig</i>} mutants (C and D) at E9.5. The YFP fate-mapped cells were identified by GFP immunostaining (green) and neurogenic placode cells were identified by TUJ1 staining (red) in the opthalamic (A and C) and facial nerve (B and D) region. The section planes in A-D are the same as those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120821#pone.0120821.g003" target="_blank">Fig. 3B,C,F and G</a>. <i>Ptch1</i>^{<i>Wig/Wig</i>} mutants (C and D) displayed much less neural crest cell (green) admixture within the ophthalmic and geniculate placodes relative to controls (A and B). Scale bars: 20μm (A and C); 50μm (B and D).</p

Generation of a novel Patched1 mutation by ENU mutagenesis.

<p>(A) Schematic depicting the <i>Patched1</i> (<i>Ptch1</i>) genomic structure indicating an A to T substitution in the 3’ region of intron 15, creating a new consensus splice acceptor site. The mutation is predicted to creates a 7 base pair insertion (box) in the 5’ end of Exon 16 that leads to a premature stop (TGA, box) 17 downstream. The mutation is referred to as <i>Wiggable</i> (or <i>Ptch1</i>^<i>Wig</i>). (B) Western blot of Hek293T cells overexpressing either wild type PTCH1 or <i>Wig</i> PTCH1 and probed with a PTCH1 amino (N)-terminal specific antibody. Wild type PTCH1 migrates as a complex centered at 170kDa. The <i>Wig</i> PTCH1 migrates at approximately 90kDa. (C-D) Whole mount images of wild type (C) and <i>Ptch1</i>^{<i>Wig/Wig</i>} (D) embryos at E11.5, which display defects in craniofacial and neural development. (E) <i>LacZ</i> staining in a <i>Ptch1</i>^{<i>LacZ/+</i>} heterozygote control showing <i>Ptch1</i> gene activity in ventral neural tissues and in endoderm. <i>Ptch1</i>^{<i>LacZ/+</i>} display normal embryonic development (F) Complementation of the <i>Ptch1</i>^<i>Lac</i>Z allele with the <i>Ptch1</i>^<i>Wig</i> (<i>Ptch1</i>^{<i>LacZ/Wig</i>}) allele, leading to open neural tube and craniofacial defects and an upregulation of <i>Ptch1</i>^<i>LacZ</i> gene activity.</p

Enhanced Shh signaling in <i>Ptch1</i>^{<i>Wig/Wig</i>} mutants affects cranial placode development.

<p>(A and D) Immunostaining of PAX3 (green), SOX10 (red), and DAPI (blue) in wild type (A) and <i>Ptch1</i>^<i>Wig</i> mutants (D). PAX3-positive cells (placodal cells) and PAX3/SOX10 double-positive cells (neural crest cells) are identified by green and white arrowheads respectively. (B, C, E and F) <i>Pax3</i> mRNA expression in control (B and C) vs. <i>Ptch1</i>^<i>Wig</i> mutants (E and F). Sections in (C and F) correspond to the dotted line in (B and E). Red arrowheads indicate reduced <i>Pax3</i> expression in <i>Ptch1</i>^{<i>Wig/Wig</i>} mutant placodal primordia (E, F vs. B, C). (G-L) Whole mount <i>in situ</i> hybridization of indicated placodal markers <i>Ngn1</i> (G and J), <i>Ngn2</i> (H and K), and <i>NeuroD1</i> (I and L) on E9.5 wild type (G-I) vs. <i>Ptch1</i>^<i>Wig</i> mutants (J-L). Red and white arrowheads identify the trigeminal and epibranchial placodes respectively. Scale bars: 20μm (A and D); 100μm (B,E and G-L); 50μm (C and F).</p

Restored <i>TOPgal</i> activity and cranial nerve development in <i>Hhat</i>^{<i>Creface/Creface</i>}; <i>Ptch1</i>^{<i>Wig/Wig</i>} double mutants.

<p>(A-C) <i>TOPgal</i> expression in E9.5 controls (A), <i>Ptch1</i>^{<i>Wig/Wig</i>} mutant (B), and <i>Hhat</i>^{<i>Creface/Creface</i>};<i>Ptch1</i>^{<i>Wig/Wig</i>} compound mutant (C) embryos. Red arrowheads indicate trigeminal nerve region and white arrowheads show the facial nerve region. (D-F) Whole mount <i>Sox10 in situ</i> hybridization in the indicated control or mutant embryos. (C and F) <i>Hhat</i>^{<i>Creface/Creface</i>};<i>Ptch1</i>^{<i>Wig/Wig</i>} double mutants showed a restoration of cranial nerve patterning indicated by <i>TOPgal</i> and <i>Sox10</i> staining levels similar to controls (A and D). Scale bars: 200μm.</p